Noxious chemical, thermal and mechanical stimuli excite peripheral nerve endings of small diameter sensory neurons (nociceptors) in sensory ganglia (eg., dorsal root, nodose and trigeminal ganglia) and initiate signals that are perceived as pain. These neurons are crucial for the detection of harmful or potentially harmful stimuli (for example heat), tissue damage caused by local tissue acidosis, and physical movement (for example tissue stretch) that arise from changes in the extracellular space during inflammatory or ischaemic conditions (Wall and Melzack, 1994).
Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the main pungent ingredient in xe2x80x9chotxe2x80x9d capsicum peppers, and its analogs interact at specific membrane recognition sites called vanilloid receptors. These receptors are expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflammation (Bevan and Szolcsanyi, 1990). Capsaicin is a very selective activator of thinly or unmyelinated nociceptive afferents (Szolcsanyi, 1993; Szolcsanyi, 1996). Capsaicin can be blocked by a selective antagonist, capsazepine. Another ligand is the potent tricyclic diterpene resiniferatoxin (RTX), (Szolcsanyi et al., 1991), a molecule that binds with nanomolar affinity at the capsaicin-binding site.
Recently, one receptor for capsaicin (VR1) was cloned from rat (Caterina et al., 1997) and shown to be a coincidence detector for H+ (low pH) and heat (Tominaga et al., 1998). VR1 is expressed in small nociceptive neurons of the dorsal root ganglion, consistent with its role in modulating peripheral pain (Tominaga et al., 1998). VR1 is a ligand-gated non-selective cation channel that shows pronounced outward rectification (Caterina et al., 1997). The vanilloid (xe2x80x9ccapsaicinxe2x80x9d) receptor VR1 is activated by capsaicin and RTX, and activation of VR1 is blocked by the antagonists capsazepine (CPZ); (Bevan et al., 1992) and ruthenium red (RR; (Wood et al., 1988)). Recently, rat VR1 and VR2 and a partial cDNA sequence of human sequences were disclosed in the WIPO publication WO 99/09140.
The densities of VR1 receptors can be tested using a [3H]RTX binding assay (Szallasi and Blumberg, 1990; Szallasi and Blumberg, 1993). Indeed, high expression of VR1 receptors was observed in rat and human spinal cord and dorsal root ganglia (Szallasi et al, 1993; Szallasi and Goso, 1994; Acs et al., 1994). Protons inhibited [3H]RTX binding to VR1 receptors (Szallasi et al. 1995).
Prior ligand binding assays using the VR-1 receptor teach that the pH must be near physiological conditions. In these assays, ligand binding was reduced by 50% and 70% at pH 8.0 and pH 9.0, respectively (Szallasi and Blumberg, 1993).
In contrast to what is suggested in the art, the present invention provides the surprising discovery that the binding capacity of certain ligands of the Vanilloid receptor increases at pH values that are greater than pH 7.4. The present invention provides improved assays to measure competitive vanilloid receptor binding of a known radiolabeled ligand and a test compound binding in aqueous buffers at a pH in the range of about 7.5-10.0. The present invention also provides the discovery that divalent cations also increase the binding capacity of certain ligands for the Vanilloid receptor. Therefore the aqueous solutions used for the methods of the present invention advantageously may include, as one component, a divalent cation.
The methods of the present invention are useful to find compounds that bind to Vanilloid receptors.